We have previously shown that nonobese diabetic (NOD) mice are selectively deficient in α/β-T cell receptor (TCR)+CD4−CD8− NKT cells, a defect that may contribute to their susceptibility to the spontaneous development of insulin-dependent diabetes mellitus (IDDM). The role of NKT cells in protection from IDDM in NOD mice was studied by the infusion of thymocyte subsets into young female NOD mice. A single intravenous injection of 106 CD4−/lowCD8− or CD4−CD8− thymocytes from female (BALB/c × NOD)F1 donors protected intact NOD mice from the spontaneous onset of clinical IDDM. Insulitis was still present in some recipient mice, although the cell infiltrates were principally periductal and periislet, rather than the intraislet pattern characteristic of insulitis in unmanipulated NOD mice. Protection was not associated with the induction of "allogenic tolerance" or systemic autoimmunity. Accelerated IDDM occurs after injection of splenocytes from NOD donors into irradiated adult NOD recipients. When α/β-TCR+ and α/β-TCR− subsets of CD4−CD8− thymocytes were transferred with diabetogenic splenocytes and compared for their ability to prevent the development of IDDM in irradiated adult recipients, only the α/β-TCR+ population was protective, confirming that NKT cells were responsible for this activity. The protective effect in the induced model of IDDM was neutralized by anti-IL-4 and anti-IL-10 monoclonal antibodies in vivo, indicating a role for at least one of these cytokines in NKT cell-mediated protection. These results have significant implications for the pathogenesis and potential prevention of IDDM in humans.